Diagnosis of rupture of the amniotic membranes prior to the onset of uterine contractions remains one of the significant clinical problems in modern obstetrics because there is not a reliable clinical method for the detection of amniotic fluid in the vagina (Gregg, (1992), Obstet. Gynecol. Clin. North Am., 19: p 241; Malee, (1992), Obstet. Gynecol. Clin. North Am., 19: p 309; Zlatnic, (1992), Obstet. Gynecol. Clin. North Am., 19: p 353; Davidson, (1991), Clin. Obstet. Gynecol., 34: p 715).
Pre-labour rupture of the fetal membranes occurs in 6.6 to 13.9% out of all patients and is followed by spontaneous labour within 48 hours in 70 to 90% of cases (Sacks et al., (1967), Am. J. Obstet. Gynecol. 83: p 930; Eastman et al., (1961), Williams Obstetrics, Appleton-Century-Crofts, New York pp. 432; Lebherz et al., (1963), Am. J. Obstet. Gynecol., 87: p 218). Pre-labour rupture of the amniotic membranes is directly or indirectly responsible for 4.5-14% of premature births and for significant mortality and morbidity associated with preterm delivery (Friedman et al., (1969), Am. J. Obstet. Hynecol. 104: p 544); Gibbs et al., (1982), Obstet. Gynecol. 60: p 671). The risks of fetal and maternal infection that accompany expectant management has to be balanced against the improvement in neonatal outcome that arises with greater maturity of the fetal organ systems. The reported latent period between rupture of the membranes and the birth varies between 12 hours and 77 days. Approximately 41% of these women will deliver within a week (Dowd et al., (1992), Aust. NZ. J. Obstet. Gynaecol. 32: p 120). Conservative management that leads to prolonging the pregnancy provides the fetus with the greatest potential for survival as outcome reflects gestational age at birth. However, pulmonary hypoplasia and infection may occur with prolonged leakage leading to oligohydramnios (Levine et al., (1991), Nurse Res., 40: p 36; Mercer supra; Morales et al., (1993), Am. J. Obstet. Gynecol. 168: p 503; Harstad et al., (1993), Am. J. Perinatol. 10: p 8).
In separate studies of 109 and 111 pregnancies respectively studied neonatal outcomes following premature, pre-labour or prolonged rupture of the membranes (PROM) at or before 34 weeks showed an overall mortality of 16.8-26.6% and morbidity of 68.8% (Airede, (1992), Ann. Trop. Paediatr. 12: p 283; Dale et al., (1989), Eur. J. Obstet. Gynecol. Reprod. Biol. 30: p 257). The occurrence of PROM at or less than 34 weeks was associated with the following complications:                prematurity        chorioamnionitis        neonatal sepsis        cardiorespiratory depression at birth        respiratory distress syndrome (RDS), and        intraventricular haemorrhage        (See, for example, Gjerdingen (1992), J. Am. Board Fam. Pract. 5: p 601; Johnson et al., (1981), Obstet. Gynecol. 57: p 547; Pasweg et al., (1992), Gynakol. Geburtshilfliche Rundsch. 32: p 222; Kilbride et al., (1989), Clin. Perinatol. 16: p 863).        
Some factors that are frequently associated with the risk of infection are gestational age, elapsed time between rupture and delivery, and socioeconomic status of the mother. Perinatal mortality rate increases two-fold after a latent period of 24 hours, doubles again within 48 hours, and after 14 days is ten times the expected rate (Overstreet et al., (1966), Am. J. Obstet. Gynecol., 96: p 1037; Major, supra). Similarly, maternal morbidity and mortality in certain populations are considerably increased with an extended latent period between rupture of the membranes and delivery (Neuhaus, 1956 and Eastman, supra).
The establishment of a simple and accurate method for diagnosis of premature rupture of the membranes has been a highly desirable outcome for sometime. In some cases the diagnosis of PROM is obvious from the sudden release of clear amniotic fluid from the vagina and its continued presence thereafter. In other cases PROM is less obvious, and it can be difficult to differentiate between the leakages of amniotic fluid from the presence of urine or endocervical mucus in the vagina.
There are currently a number of non-invasive procedures for diagnosing PROM. These include:                Identification of fetal squamous cells or hairs (Phillip et al., (1929), Zentrabl. Gynakol. 26: p 1618);        Microscopy for the identification of fetal fat globules within or outside fetal cells following staining (Friedman et al., (1969), Am. J. Obstet. Gynecol. 104: p 544;        Estimation of vaginal fluid pH (Nitrazine paper test) (Minkoff et al., (1987), Am. J. Obstet. Gynecol. 150: p 965; International Patent Application No. WO02/054949; U.S. Pat. No. 4,357,945);        Amniotic fluid crystallization patterns following desiccation (Gorodeski et al., (1982), J. Perinat. Med. 10: p 286; Reece et al., (1984), Obstet. Gynecol. 64: p 248; Brookes et al., (1986), Aust. NZ. J. obstet. Gynecol. 26: p 160);        Amniotic fluid heating (Dalkalitsis et al., (1989), Z. Geburtshuilfe. Perinatol. 193: p 183);        Measurement of the activity of diamine oxidase in vaginal fluid (Gahl et al., (1982), Obstet. Gynaecol. 60: p 297);        Prolactin in vaginal fluid (Huber et al., (1983), Br. J. Obstet. Gynaecol. 90: p 1183);        Alpha feto-protein in vaginal fluid (Garite, (1990), Am. J. Obstet. Gynecol. 151: p 1001);        Insulin-like growth factor binding protein-1 in vaginal fluid (Rutanen et al., (1993), Clin. Chim. Acta. 214: p 73; Woltmann et al., (1995), Z. Geburtshilfe. Neonat. 199: p 243; Darj et al., (1998), Acta. Obstet. Gynecol. Scand. 77: p 295; Ragosch et al., (1996), Geburtshilfe Fravenheilkd. 56: p 291; U.S. Pat. Nos. 5,554,504); 5,597,700);        Human placental lactogen in vaginal fluid Huber et al., (1983), Br. J. Obstet. Gynaecol. 90: p 1183;        Colorimetric detection (bromothymol blue) (GB Patent No. 2,353,357);        Measurement of inhibin B (International Patent Application No. WO98/59245);        Fetal fibronectin in vaginal fluid (Rutanen supra; Ragosch supra; Trovo et al., (1998), Minerva Ginecol. 50: p 519); and        PROM-Protein, (International Patent Application No. WO94/21687).        
However, while all of these procedures provide some indication that PROM has taken place they are not completely accurate. In the most part, previously investigated markers for diagnosing PROM provided false positive results owing to the presence of interfering substances. Such substances for the most part are common and which occur in the vagina, examples of these are urine, meconium, pregnancy serum and blood. In many instances, the presence of blood is responsible for a false positive as the markers tested are also found at varying levels in maternal blood (Huber J et al., 1981, Br J Obstet Gynaecol, 57:547; Phocas et al., 1989, Eur J Obstet Gynecol Reprod Biol., 31:133; Broe et al., 1992, Clin Chem., 38:784; Hellemans et al., 1992, Eur J Obstet Gynecol Reprod Biol., 43:173.
The inventor has now developed a simple method of determining whether or not amniotic fluid is released prematurely, which is non-invasive and accurate.